The present invention relates generally to an apparatus and method for rapid and sensitive detection of contaminants in liquids, and more particularly to a biocompatible apparatus and method for detecting and measuring low levels of bacteria.
There are many industries in which it is necessary to detect various contaminants in liquids and liquid streams. For instance, in the course of beverage processing, the beverage may become contaminated with bacteria. Once contaminated, food poisoning or spoilage often occurs. Moreover, pathogenic organisms may exist in the product and cause harm to consumers after they use or consume the product. Thus, the ability to monitor and detect the level of contaminants in liquids is critical for maintaining a product that can be consumed and/or used by humans.
As another example, it is necessary to monitor and detect the level of bacteria in the production process for beer. More specifically, undue levels of bacteria in beer, typically Pectinatus cerevisiiphilus, can lead to spoilage and the like where pasteurization is not utilized in the production process. The system of detection needs to be rapid and sensitive while being cost-effective. Additionally, such a detection system should desirably be automated or semi-automated so as not to be unduly labor intensive, and be configured for connection to process control systems.
Unfortunately, prior art methods for detecting bacteria contamination are too cumbersome and time consuming for immediate use. Currently, it is believed that culturing methods are being utilized. Such methods are extremely slow (requiring up to several days or so) and are very labor intensive.
One type of detection method captures the bacteria being detected on a filter medium. Detection is then achieved with chemiluminescence and fluorescence imaging. While detection can be carried out in about one hour or so and the detection-sensitivity is approximately one bacterial event in the sample, this system has no mechanism to achieve selectivity, is highly labor intensive and relatively expensive.
Further, it has been proposed to utilize immunoabsorbents and electrochemistry so as to achieve immunochemical detection. Such a system is not believed to be either automated or have suitable detection sensitivity.
It has also been proposed to employ DNA-hybridization and/or polymerase chain reaction (PCR) methods. Such methods are highly labor intensive and very dependent upon the skill of the operator.
Additionally, there has been considerable attention directed to the general area of flow cytometers and particle analysis using sheath flow chambers with laser excitation and detection of fluorescence using photodetectors. The purpose of such sheath flow chambers is to entrain the sample solution in a fast moving stream of pure water or buffer such that the sample solution is swept out of the sheath flow chamber and down into a square bore capillary. U.S. Pat. Nos. 3,871,770 and 4,343,551 disclose sheath flow chamber designs.
Many flow cytometers impinge the sample into a flat glass sheet, by way of, for example, a microscope cover slip. This forces the cells in the flow stream to experience a shear field. For mammalian cells, which are oblate spheroids, this causes such cells to align in the shear field with their short axis along the surface normal of the cover slip (see, for example, U.S. Pat. No. 4,988,619). A laser beam then impinges along the surface normal, which maximizes the scattering signal that is used to trigger the fluorescence detection channels. However, this system causes a very large amount of scattering that contributes significantly to the background signal.
U.S. Pat. No. 4,983,038 discloses one design using flow cytometry. U.S. Pat. No. 4,660,971 discloses one approach for stabilizing the optical arrangement of a sheath flow and collection system.
Another drawback with these and other flow cytometer designs is that they use glass or stainless steel components, which can adsorb bacteria, organisms or other contaminants that are to be detected. When detecting high levels of bacteria, organisms or other contaminants, this is not a serious issue. However, when attempting to detect low levels of bacteria, organisms or other contaminants, the loss of a few organisms to adsorption on the walls of the delivery tubing or other components can lead to the drastic reduction in sensitivity.
The problem with measuring low bacteria levels, for instance, is that bacteria adsorb tenaciously to many, if not most surfaces. The adsorption is accomplished in several ways: through electrostatic interactions (e.g., negatively charged bacteria adsorb to a positively charged surface) and/or hydrophobic interactions. Many bacteria also exude complex polysaccharides called exopolysaccharides (EPS). These are large hydrophobic polymeric molecules that are strongly adsorbed to many surfaces, and thereby provide an anchor point for bacteria. Several EPS types are known; some are charged. Positively charged EPS allows negatively charged bacteria to adsorb to positively charged surfaces by providing an electrostatic buffer (e.g., positive EPS adsorbs to negative surface, then negative bacterium adsorb to the positively charged EPS surface).
The tendency for bacterial adsorption at surfaces makes the detection of small numbers of bacteria difficult, primarily due to the inefficient delivery of the bacteria to the detection region. It is generally assumed that if the bacteria are actually delivered to the detection region, then they will probably be detected with little difficulty. However, as explained above, this is not the case with conventional cytometers. Rather, conventional cytometer designs using glass and/or stainless steel components are not compatible with the measurement of low levels of bacteria.
In addition, conventional flow cytometers run at very high sheath flow rates. The high sheath flow rate translates into the bacteria passing through the detection site at a very rapid rate, thus the bacteria spend a short time in the laser beam. Consequently, there are very few fluorescent photons produced for detection. For mammalian cells (which are 10,000xc3x97 larger in volume than typical bacteria cells) this is not a serious issue, because their larger size means their surfaces can accommodate more label. Since there is more label present on the surfaces of the mammalian cells, the sample can run faster while still allowing for the collection of a sufficient number of fluorescent photons to detect them efficiently. However, for the much smaller bacterial cells, the high flow rates are not compatible for measuring fluorescence from such cells.
In summary, despite the considerable prior efforts in this field, there remains the need for a versatile, rapid and sensitive, cost-effective, biocompatible detection system for small levels of bacteria, organisms and other contaminants or moieties of interest.
Accordingly, the present invention provides a rapid and sensitive detection system that is capable of automation. Moreover, the invention can be utilized in a cost-effective fashion to detect a variety of contaminants in many liquid streams, as well as allowing the detection of other moieties of interest by utilizing a liquid stream.
The present invention provides a biocompatible flow cytometry system such that the bacteria or other particles in the sample will not adsorb onto the system""s surfaces. This is accomplished by fitting the system with biocompatible material.
The present invention provides a system characterized by ready and easy alignment of the laser source with the sample stream. In addition, the invention provides a system capable of maximizing the signal emitting from the contaminant or other moiety being detected while minimizing the background signal, i.e., providing a system having a desirable signal/noise ratio. A principal embodiment of the invention is a system in which the detection signal is within the red region of the light spectrum.
The present invention provides a system that utilizes a flow chamber system that essentially eliminates background scattering, includes a holder which is sufficiently mechanically rugged so as to accommodate a relatively frail, thin wall square bore capillary and allows facile removal and replacement of such capillary with minimal realignment of the optical components of the system.
This description will be principally directed to the rapid detection of beer spoilage bacteria. However, it is readily appreciated by one of ordinary skill in the art that the method and apparatus described and illustrated can be employed to detect contaminants of other bacteria, and microorganisms. This system is particularly useful for detecting a particulate material of interest, preferably capable of holding at least 100 or so fluorophores. A wide variety of liquids and liquid streams in the fermentation, food and dairy industries (e.g., beverages and juices) require such detection and/or monitoring; and the present invention can be utilized for such applications.
Even further, while the more usual applications for the invention will involve the detection and/or monitoring of bacteria or the like already present in a liquid or liquid stream (e.g., beer), it should be appreciated that the invention can also be utilized in applications where it is necessary to first provide the liquid so as to capture any contaminant to be detected, for example, any technique where a vessel or environment is used for chemical or biochemical synthesis that needs to be monitored to insure the absence, or relative absence, of unwanted contaminants that would in some way hinder the synthesis. In addition, the system of this invention may be utilized to detect the level of any moiety of interest. For example, soil samples could be added to water or the like; and a moiety of interest could be detected. Additionally, this method could be used to detect the level of E. coli in meat or the like.
Other features and advantages of the present invention will become apparent from the following detailed description and drawings. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.